Background: Many different walking capacity test formats are being used. It is unclear whether walking speed, obtained from short tests, and walking distance, obtained from long tests, provide different clinical information. Objectives: To determine the differential effect of various short and long walk test formats on gait velocity, and the actual relationship between walking speed and walking distance in multiple sclerosis (MS) patients with diverse ambulation status. Methods: A cross-sectional multicentre study design was applied. Ambulatory MS patients (Expanded Disability Status Scale (EDSS) 0-6.5; n = 189) were tested at 11 sites. Short tests consisted of the Timed 25-Foot Walk (static start, fastest speed) and 10-Metre Walk Test (dynamic start, usual and fastest speed). Long tests consisted of the 2- and 6-Minute Walk Tests (fastest speed). Subjects were divided into mild (EDSS 0-4; n = 99) or moderate (EDSS 4.5-6.5; n = 79) disability subgroups. Results: In both subgroups, the start protocol, instructed pace and length of test led to significantly different gait velocities. Fastest walking speed and 6-Minute walking distance showed the strongest correlation (R-2 = 0.78 in mild and R-2 = 0.81 in moderate MS; p < 0.01). Short tests' relative estimation errors for 6-Minute walking distance were 8-12% in mildly and 15-16% in moderately affected subjects. Based on the 2-Minute Walk Test, estimation errors significantly reduced to approximately 5% in both subgroups. Conclusions: A single short test format at fastest speed accurately describes an MS patient's general walking capacity. For intervention studies, a long test is to be considered. We propose the Timed 25-Foot Walk and 2-Minute Walk Test as standards. Further research on responsiveness is needed.